Glaucoma is one of the most common causes of blindness worldwide and, while there are many different forms of glaucoma that differ significantly in clinical presentation and disease progression, they all share a common endpoint which is the loss of retinal ganglion cells (RGCs). New treatments are needed, since current therapies can delay, but not stop disease progression. One of the major barriers to the development of novel treatments is the incomplete understanding of the disease pathogenesis. Recent evidence indicates the loss of RGCs is due to apoptosis, nevertheless, the actual molecular mechanism that triggers apoptosis is still controversial. Fas Ligand (FasL) is a pro-apoptotic protein that is constitutively expressed in the retina where it is thought to protect tissue from destruction by maintaining immune privilege, either by inducing apoptosis of infiltrating inflammatory cells or by preventing neoangeogenesis. However, there are two forms of FasL, a membrane-bound form (mFasL) and a soluble form (sFasL) that is produced by metalloproteinase cleavage of the membrane-bound protein. Our previous studies on the function of mFasL and sFasL indicate that mFasL is pro-apoptotic, while sFasL is anti-apoptotic. We recently developed a unique knock-in mouse strain in which the FasL metalloproteinase cleavage sites were mutated to prevent cleavage of the membrane-bound protein. In these mice, FasL is expressed and regulated normally, but they are unable to cleave FasL and therefore, can only express mFasL (termed ? CS mice). For the first time, these mice have allowed us to study the in vivo function of membrane FasL in the absence of soluble FasL. In order to determine the function of mFasL in the development of glaucoma, we backcrossed the ? CS knock-in mutation into the DBA/2J strain (D2. ? CS mice) that develops spontaneous elevated intraocular pressure (IOP) and loss of RGCs. Our preliminary results indicate that mFasL is a critical mediator of RGC apoptosis during the development of glaucoma. Moreover, sFasL has an important neuroprotective effect that not only prevents loss of RGCs, but also protects other retinal neurons in hypertensive eyes. We hypothesize that in response to injury caused by elevated IOP, there is increased expression of Fas and FasL within the retina. However, the extent of retinal apoptosis is determined by the ratio of membrane / soluble FasL expressed by microglia. Neurotoxic microglia express higher levels of mFasL, while neuroprotective microglia express higher levels of sFasL. This hypothesis will be tested in three Specific Aims: (Aim 1) Demonstrate that elevated IOP in D2. ? CS mice that are unable to cleave FasL triggers increased retinal neurotoxicity. (Aim 2) Determine whether increased retinal neurotoxicity in D2. ? CS mice is due to increased mFasL expression and/or the absence of sFasL. (Aim 3) Determine the mechanisms of microglia-mediated neurotoxicity in vitro using a co-culture system with immortalized retinal microglia from D2. ? CS mice and primary RGCs. PUBLIC HEALTH RELEVANCE: Glaucoma is a chronic disease that can ultimately lead to loss of vision and blindness. The loss of vision occurs when a certain type of cell in your retina dies (retinal ganglion cells). It is unclear to scientists exactly why these cells die. We have discovered at least one reason why these cell die. In addition, we have also found a way to stop these cells from dying. This research will lead to novel targets for therapeutic intervention in glaucoma.